STORM is the most widely used SRM method, and it relies on the sequential activation and time-resolved localization of photoswitchable fluorophores to generate high-resolution images. To achieve a high-quality multicolor image, specific labeling is required using direct labeling, protein conjugates, or antibody staining. Ideal fluorophores for STORM should be very bright and have a high rate of photoswitching cycles. They should also exhibit minimal photobleaching in thiol-containing buffers. With appropriate dye and buffer combinations, an optimized STORM system can generate images with 5 nm resolution. Numerous publications show combinations of Molecular Probes® dyes with a variety of targeting and labeling specificities used to generate multiplex STORM images.

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Antibody conjugates and labels

Molecular Probes® fluorophores have been tested in STORM applications, as conjugates of antibodies, proteins, dextrans, and other biomolecules. Use the table below to select the fluorophore with the best wavelength and rating for your application in either dSTORM or nSTORM—citations are listed for each. By following the links you can also find all of the bioconjugates of each fluorophore currently available to target your molecule of interest; the list ranges from antibodies and phalloidins to growth factors and lectins. If you don't find the conjugate you need on the list, the links will also show you reactive dye forms or optimized labeling kits to enable you to create your own bioconjugate probes.

Nucleic acid stains

For direct labeling of nucleic acids, SYTO® 13 and YOYO®-1 dyes have demonstrated excellent performance in STORM applications. Both dyes exhibit green fluorescence and are imaged using FITC filters for conventional applications. One of the highest affinity nucleic acid stains, the cell-impermeant YOYO®-1 stain exhibits over a thousand-fold increase in fluorescence when bound to dsDNA. YOYO®-1 stain has proved extremely useful in the analysis of single molecules of DNA. SYTO® 13 dye is cell permeant and shows a small shift in fluorescence characteristics (excitation and emission maxima) when bound to RNA vs. DNA. Use the table below to select a DNA dye for your STORM experiment.

Organelle probes

Direct labeling probes for a range of organelles have been implemented in STORM with outstanding results. For mitochondrial labeling the optimal STORM signal is obtained with MitoTracker® Red dye, although MitoTracker® Orange and MitoTracker® Deep Red both offer good performance if the Texas Red® dye channel is already in use for multiplexing another signal.

LysoTracker® Red is recommended for lysosomes and other acidic organelles, and ERTracker® Red is highly selective for the endoplasmic reticulum.

The plasma membrane can be labeled for STORM with a range of dyes offering orange- (DiI), far-red– (DiD), or near-IR– (DiR) fluorescence. All of these dyes offer good performance in STORM, but DiD offers the strongest performance if the far-red channel is available. Use the table below to select appropriate organelle probes for your STORM experiment.

Buffers and reagents

To enable effective photoswitching, fluorescent dyes require either an appropriate activator/reporter pairing (nSTORM) or an oxygen-scavenging buffer system (dSTORM). Typical buffer systems contain catalase, glucose, and glucose oxidase (commonly referred to as GLOX) in combination with a reducing agent. The two most commonly used are mercaptoethylamine (MEA) or β-mercaptoethanol (BME). Both xanthene- and cyanine-based dyes can be used in either buffer system, however xanthene-based dyes (Alexa Fluor® 488 and Alexa Fluor® 568) tend to perform better in MEA whereas cyanine-based dyes (Cy®5, Alexa Fluor® 647, and Alexa Fluor® 750) perform better in BME and TCEP, respectively. See the buffer composition and references in the table below. Use this osmotically-stabilized imaging buffer as a starting point and optimize for a particular dye using the different reducing agents.